1. Field of the Invention
The present invention relates to a method for preparing closed vesicles which comprises the step of rehydrating closed vesicles composed of dehydrated micelle particles or dehydrated amphipathic micelle bilayers under a specific condition. The present invention also relates to closed vesicles obtained by said method.
2. Background Art
Liposomes are closed vesicles composed of lipid micelle bilayers. They can load fat-soluble substances in the lipid phase and water-soluble substances in the aqueous phase, and can carry macromolecular substances such as proteins as well as low-molecular compounds. They also have high bio-compatibility. For these reasons, various research has been made focusing on their use as carriers of drugs, proteins, nucleic acids and the like for the drug delivery systems (DDS). In recent years, practical research has also progressed particularly on liposomes provided with targeting function through a surface modification of the liposome, in addition to reduction of toxicity and improvement of blood retention time by loading a drug.
However, micelle structures of liposomes are essentially not sufficiently stable from a viewpoint of thermodynamics, and development of methods for long term storage is absolutely necessary for their practical uses.
Japanese Patent Unexamined Publication (KOKAI) No. (Sho) 53-142514/1978 to Evans et al. discloses a xe2x80x9cdehydration methodxe2x80x9d aiming at long-term stability which comprises steps of removing water from a liposome solution to convert into a stable dried lipid state, followed by adding an aqueous solution before use (rehydration) to regenerate liposomes. This method suggested a possibility of long term storage of liposomes in the form of dried powder. Since then, research to achieve improvements has been conducted in order to reduce aggregation, fusion of liposomes, and leaking of loaded substances upon the dehydration.
For example, from viewpoints of protective agents for dehydration, Japanese Patent Publication (KOKOKU) No. (Sho) 61-21449/1986 discloses a method comprising a step of lyophilizing a liposome solution after the addition of hydrophilic compound such as dextran or gum arabic, and Japanese Patent Unexamined Publication for PCT Application (KOHYO) No. (Sho) 62-501631/1987 discloses a method comprising a step of lyophilizing a liposome solution containing a disaccharide such as trehalose or sucrose. In addition, Japanese Patent Unexamined Publication (KOKAI) No. (Hei) 7-145041/1995 discloses liposomes loading an antitumor agent which is dehydrated by a method other than lyophilization process, and Japanese Patent Unexamined Publication (KOKAI) No. (Hei) 7-145043/1995 discloses a method comprising a step of dehydrating a liposome solution without pre-freezing and maintaining a residual water content of 2 to 5%.
Furthermore, from a viewpoint of a solution for the rehydration, Japanese Patent Unexamined Publication (KOKAI) No. (Sho) 53-142514/1978 discloses that lyophilized liposomes were dispersed under warming at a temperature of 50 to 70xc2x0 C.
However, leaking of loaded substances in a range of about tens to 10% were observed with these methods depending on lipid compositions and the type of loaded substances, and stability of liposomes is sometimes insufficient.
Such leaking of-loaded substance may cause significant disadvantages, in particular, as for liposomes used for assays which can be significantly influenced by a leak of a loaded agent, or liposomes containing a medicament having a potent physiological activity such as an anti-tumor agent or the like. When liposomes loading an anti-tumor agent are clinically applied, even a leak of an amount of 10-30% cannot be a negligible problem because it may affect a reduction of side effects achieved by a liposome formation, or may possibly deteriorate targeting functions.
The inventors of the present invention conducted research to solve the aforementioned problems of the state of the art. As a result, they found that conditions for the rehydration are unexpectedly important as well as processes having been studied so far, and that the temperature of a dispersion at rehydration is significantly influential. It was found quite surprisingly that the leak of the loaded substances is remarkably suppressed by carrying out the rehydration under cooling, contrary to the conventional warming process for closed vesicles including liposomes, and that closed vesicles rehydrated under a low temperature condition have highly improved stability even after they are warmed up to room temperature. The present invention was achieved on the basis of these findings.
The present invention thus provides a method for preparing a closed vesicle by a rehydration of a closed vesicle comprising a dehydrated micelle particle or dehydrated amphipathic micelle bilayers by using a rehydration solution, characterized in that the rehydration is carried out at a low temperature.
According to preferred embodiments of the present invention, there are provided the aforementioned method wherein the temperature is in the range of from 0xc2x0 C. to 10xc2x0 C.; the aforementioned method wherein the closed vesicle is introduced with a pharmaceutically active substance or a diagnostic agent; the aforementioned method wherein the pharmaceutically active substance is an anti-tumor agent; the aforementioned method wherein the anti-tumor agent is selected from the group consisting of adriamycin, daunomycin, vinblastine, and pharmaceutically acceptable salts and derivatives thereof; the aforementioned method wherein the pharmaceutically acceptable salts are formed together with a multivalent anionic substance; the aforementioned method wherein the multivalent anionic substance is selected from the group consisting of citric acid, tartaric acid, and glutamic acid; the aforementioned method wherein the closed vesicle is a liposome; the aforementioned method wherein the liposome is obtained by lyophilizing or spray-drying a liposome containing a saccharide; the aforementioned method wherein the liposome is modified with an antibody and/or polyethylene glycol; and the aforementioned method wherein the rehydration solution has a pH within a neutral area.
According to further aspects of the present invention, there are provided a closed vesicle obtainable by any one of the aforementioned methods; and a pharmaceutical composition containing the above-defined vesicle.
According to still further aspects of the present invention, there are provided a pharmaceutical kit which comprises a dehydrated preparation comprising the dehydrated closed vesicle and a rehydration solution for rehydrating said closed vesicle, characterized in that either or both of the dehydrated preparation and the rehydration solution are used at a low temperature; and a diagnostic kit which comprises a dehydrated preparation comprising the dehydrated closed vesicle and a rehydration solution for rehydrating said closed vesicle, characterized in that either or both of the dehydrated preparation and the rehydration solution are used at a low temperature.